A sandwich enzyme-linked immunosorbent assay for adducts of polycyclic aromatic hydrocarbons with human serum albumin

Adducts of benzo[a]pyrene-diolepoxide (BPDE) with blood nucleophiles have been used as biomarkers of exposure to polycyclic aromatic hydrocarbons (PAHs). The most popular such assay is a competitive enzyme-linked immunosorbent assay (ELISA) that employs monoclonal antibody 8E11 to detect benzo[a]pyrene tetrols following hydrolysis of BPDE adducts from lymphocyte DNA or human serum albumin (HSA). Here we used 8E11 as the capture antibody in a sandwich ELISA to detect BPDE-HSA adducts directly in 1-mg samples of HSA or 20 μl of serum/plasma. The assay employs an anti-HSA antibody for detection, and this is amplified by an avidin/biotinylated horseradish peroxidase complex. The sandwich ELISA has advantages of specificity and simplicity and is approximately 10 times more sensitive than the competitive ELISA. To validate the assay, HSA samples were assayed from three populations with known high PAH exposures (coke oven workers), medium PAH exposures (steel factory control workers), and low PAH exposures (volunteer subjects) (n = 30). The respective geometric mean levels of BPDE-HSA adducts—67.8, 14.7, and 1.93 ng/mg HSA (1010, 220, and 28.9 fmol BPDE equiv/mg HSA)—were significantly different (P < 0.05). The sandwich ELISA will be useful for screening PAH exposures in large epidemiologic studies and can be extended to other adducts for which capture antibodies are available.     

Excessive ingestion of long-chain polyunsaturated fatty acids during developmental stage causes strain- and sex-dependent eye abnormalities in mice

The eyes are riched in long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic acid [ARA; 20:4 (n−6)] and docosahexaenoic acid [DHA; 22:6 (n−3)]. Despite their abundance in the eyes, ARA and DHA cannot be sufficiently synthesized de novo in mammals. During gestation, eye development is exceptionally rapid, and substantial amounts of LC-PUFAs are needed to ensure proper eye development. Here, we studied the influences of dietary LC-PUFAs in dams (C57BL/6 and C3H/He) on the eye morphogenesis and organogenesis of their pups. Intriguingly, fetuses and newborn mice from C57BL/6 dams fed an LC-PUFA (particularly ARA)-enriched diet displayed a much higher incidence of eye abnormalities such as microphthalmia (small eye) and corneal opacity than those from dams fed an LC-PUFA-poor diet. The effects of LC-PUFAs on eye anomalies were evident only in the female pups of C57BL/6 inbred mice, not in those of C3H/He mice or male C57BL/6 mice. These results demonstrate a gene-by-environment (GxE) interaction in eye development in mice. Furthermore, our molecular analysis suggested the potential roles of Pitx3 and Pax6 in the above interaction involving ARA.     

One residue substitution in PcyA leads to unexpected changes in tetrapyrrole substrate binding

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the sequential reduction of the vinyl group of the D-ring and A-ring of biliverdin IXα (BV), using reducing equivalents provided by ferredoxin. This reaction produces phycocyanobilin, a pigment used for light-harvesting and light-sensing in red algae and cyanobacteria. The crystal structure of PcyA-BV reveals that BV is specifically bound in the PcyA active pocket through extensive hydrophobic and hydrophilic interactions. During the course of a mutational study of PcyA, we observed that mutation of the V225 position, apart from the processing sites, conferred an unusual property on PcyA; V225D mutant protein could bind BV and its analog BV13, but these complexes showed a distinct UV-vis absorption spectrum from that of the wild-type PcyA-BV complex. The crystal structures of BV- and BV13-bound forms of V225D protein revealed that gross structural changes occurred near the substrate-binding pocket, and that the BV/BV13 binding manner in the pocket was dramatically altered. Protein folding in V225D-BV/BV13 was more similar to that of substrate-free PcyA than that in PcyA-BV; the “induced-fit” did not occur when BV/BV13 was bound to the V225D protein. The unexpected structural change presented here provides a cautionary note about interpreting functional data derived from a mutated protein in the absence of its exact structure.     

Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenic reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-γ (PPARγ). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-κB activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression via PPARγ activation.     

Allosteric regulation of glycogen synthase controls glycogen synthesis in muscle

Glycogen synthase (GS), a key enzyme in glycogen synthesis, is activated by the allosteric stimulator glucose-6-phosphate (G6P) and by dephosphorylation through inactivation of GS kinase-3 with insulin. The relative importance of these two regulatory mechanisms in controlling GS is not established, mainly due to the complex interplay between multiple phosphorylation sites and allosteric effectors. Here we identify a residue that plays an important role in the allosteric activation of GS by G6P. We generated knockin mice in which wild-type muscle GS was replaced by a mutant that could not be activated by G6P but could still be activated normally by dephosphorylation. We demonstrate that knockin mice expressing the G6P-insensitive mutant display an ～80% reduced muscle glycogen synthesis by insulin and markedly reduced glycogen levels. Our study provides genetic evidence that allosteric activation of GS is the primary mechanism by which insulin promotes muscle glycogen accumulation in?vivo.     

Involvement of Ca2+-dependent proteasome in the degradation of both cyclin B1 and Mos during spontaneous activation of matured rat oocytes

In matured rat oocytes, spontaneous activation from the metaphase-II (MII) stage occurred after collection from the oviducts. It is well known that the mitogen-activated protein kinase (MAPK) pathway and p34(cdc2) kinase play an important role in the arrest at MII in other species. However, there is no information about the difference in these factors among strains of rats. In the present study, in spontaneously activated oocytes from the Wistar rat, the Mos protein level and the activity of MAPK kinase (MEK)/MAPK were decreased at 120 min (13.8, 25.7, and 19.3, respectively, P<0.05), whereas Sprague-Dawley (SD) oocytes, which were not spontaneously activated, had a high level of Mos protein and MEK/MAPK activity (75.9, 76.2, and 87.9, respectively, P<0.05). Phosphorylation of MAPK in the SD oocytes was significantly suppressed by MEK inhibitor, U0126 at 60 min; this treatment decreased p34(cdc2) kinase activity via cyclin B1 degradation in a time-dependent manner. The treatment with proteasome inhibitor, MG132 or Ca2+-chelator, BAPTA-AM, overcame the spontaneous degradation of both Mos and cyclin B1 in a dose-dependent manner in Wistar oocytes. More than 90% of Wistar oocytes treated with BAPTA-AM were arrested at MII until 120 min. In conclusion, SD oocytes carrying Mos/MEK/MAPK, maintained a high activity of p34(cdc2) kinase by stabilizing cyclin B1, thus involved in their meiotic arrest. In contrast, Wistar oocytes had a relatively low cytostatic factor activity; rapid decrease of Mos/MEK/MAPK failed to stabilize both cyclin B1 and Mos, and these oocytes were likely to spontaneously activate.     

A test of the effect of floral color change on pollination effectiveness using artificial inflorescences visited by bumblebees

Floral color change has been recognized as a pollination strategy, but its relative effectiveness has been evaluated insufficiently with respect to other floral traits. In this study, effects of floral color change on the visitation pattern of bumblebees were empirically assessed using artificial flowers. Four inflorescence types were postulated as strategies of flowering behavior: type 1 has no retention of old flowers, resulting in a small display size; type 2 retains old flowers without nectar production; type 3 retains old flowers with nectar; and type 4 retains color-changed old flowers without nectar. Effects of these treatments varied depending on both the total display size (single versus multiple inflorescences) and the pattern of flower-opening. In the single inflorescence experiment, a large floral display due to the retention of old flowers (types 2–4) enhanced pollinator attraction, and the number of flower visits per stay decreased with color change (type 4), suggesting a decrease in geitonogamous pollination. Type-4 plants also reduced the foraging time of bees in comparison with type-2 plants. In the multiple inflorescence experiment, the retention of old flowers did not contribute to pollinator attraction. When flowering occurred sequentially within inflorescences, type-4 plants successfully decreased the number of visits and the foraging time in comparison with type-2 plants. In contrast, floral color change did not influence the number of visits, and it extended the foraging time when flowering occurred simultaneously within inflorescences but the opening of inflorescences progressed sequentially within a plant. Therefore, the effectiveness of floral color change is highly susceptible to the display size and flowering pattern within plants, and this may limit the versatility of the color change strategy in nature.     

Biotransformation of raspberry ketone and zingerone by cultured cells of Phytolacca americana

The biotransformation of raspberry ketone and zingerone were individually investigated using cultured cells of Phytolacca americana. In addition to (2S)-4-(4-hydroxyphenyl)-2-butanol (2%), (2S)-4-(3,4-dihydroxyphenyl)-2-butanol (5%), 4-[4-(beta-d-glucopyranosyloxy)phenyl]-2-butanone (19%), 4-[(3S)-3-hydroxybutyl]phenyl-beta-d-glucopyranoside (23%), and (2S)-4-(4-hydroxyphenyl)but-2-yl-beta-d-glucopyranoside (20%), two biotransformation products, i.e., 2-hydroxy-4-[(3S)-3-hydroxybutyl]phenyl-beta-d-glucopyranoside (12%) and 2-hydroxy-5-[(3S)-3-hydroxybutyl]phenyl-beta-d-glucopyranoside (11%), were isolated from suspension cells after incubation with raspberry ketone for three days. On the other hand, two compounds, i.e., (2S)-4-(4-hydroxy-3-methoxyphenyl)but-2-yl-beta-d-glucopyranoside (17%) and (2S)-2-(beta-d-glucopyranosyloxy)-4-[4-(beta-d-glucopyranosyloxy)-3-methoxyphenyl]butane (16%), together with (2S)-4-(4-hydroxy-3-methoxyphenyl)-2-butanol (15%), 4-[4-(beta-d-glucopyranosyloxy)-3-methoxyphenyl]-2-butanone (21%), and 4-[(3S)-3-hydroxybutyl]-2-methoxyphenyl-beta-d-glucopyranoside (24%) were obtained upon addition of zingerone. Cultured cells of P. americana can reduce, and regioselectively hydroxylate and glucosylate, these food ingredients to their beta-glycosides.     

Postglacial population expansion of Japanese macaques (<Emphasis Type="Italic">Macaca fuscata</Emphasis>) inferred from mitochondrial DNA phylogeography

We investigated the diversity and phylogeography of mitochondrial DNA (mtDNA) in Japanese macaques (Macaca fuscata), an endemic species in Japan that has the northernmost distribution of any non-human primate species. DNA samples from 135 localities representing the entire range of this species were compared. A total of 53 unique haplotypes were observed for the 412-bp partial mtDNA control region sequence, with length variation distinguishing the two subspecies. Clustering analyses suggested two putative major haplogroups, of which one was geographically distributed in eastern Japan and the other in western Japan. The populations in the east showed lower mtDNA diversity than those in the west. Phylogeographical relationships of haplotypes depicted with minimum spanning network suggested differences in population structure. Population expansion was significant for the eastern but not the western population, suggesting establishment of the ancestral population was relatively long ago in the west and recent in the east. Based on fossil evidence and past climate and vegetation changes, we inferred that the postulated population expansion may have taken place after the last glacial period (after 15,000 years ago). Mitochondrial DNA showed contrasting results in both variability and phylogenetic status of local populations to those of previous studies using protein variations, particularly for populations in the periphery of the range, with special inference on habitat change during the glacial period in response to cold adaptation. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.